Separation of biomolecules from a mixture has traditionally been performed by utilizing chromatographic techniques, filtration or precipitation. The continuing surge in the development of biotechnology products and processes has brought with it the need for efficient and cost effective separation and purification processes and apparatus. The preparation of biomolecules by fermentation processes can be divided into two general categories which are generally referred to as “upstream” and “downstream” processes. The upstream processes address the biochemical design of the system to produce the desired biopharmaceutical product and the downstream processes focus on harvesting and purifying the final product.
Downstream processing typically involves (1) the release of the contents of the fermentation cells, if necessary, for example by cell disruption, since the fermentation product, e.g. a protein or polynucleotide, such as a plasmid, may already be in the culture supernatant because of secretion by the host cell; (2) centrifugation to provide clarification of the contents, typically by separating the cell debris from the mother liquor which contains the desired product and other biological entities; (3) ultrafiltration to concentrate the mother liquor for subsequent steps; and (4) final product purification, typically by liquid chromatographic techniques using multi-method separation methods, e.g., ion exchange, hydrophobic interaction, reverse phase.
Chromatography resins used for protein adsorption generally comprise macroporous, hydrophilic materials. In traditional chromatographic techniques conventional granular chromatography materials that have defined particle and pore sizes are used. Porosity is essential to provide sufficient surface area for high capacity, while hydrophilic surfaces enable reversible adsorption. Base materials for chromatographic resins are usually cross-linked natural polymers, like cellulose, dextran or agarose, as well as synthetic polymers made of polyacrylamide, polymethacrylate and polystyrene divinylbenzene derivatives. The latter is often coated with a hydrophilic polymer. Ion exchange resin, hydrophobic interaction resin or affinity resins are coupled with functional ligands by chemical derivatization or by surface grafting technologies.
In the industry, upstream manufacturing capacities have increased dramatically, with many manufacturers choosing to operate several 10000 L bioreactors simultaneously. However, standard chromatography methods do not allow rapid scale-up.
At the first stage of a chromatographic purification step (also called the capture step), due to the large sample volume that has to be processed, large bed volumes are generally used. However, large columns suffer from scale-related packing problems such as hysteresis, edge-effects and resin compression, which result in unpredictable fluid distribution and pressure drops.
The performance of packed chromatography columns in industrial and preparative applications is limited by the maximum allowable pressure drop. Due to the pressure drop restrictions, resin beads with larger diameter such as larger than 40 μm are used.
The capturing of the biomolecules relies on pore diffusion, but large biomolecules do not readily diffuse into the pores, and the diffusional pathway is increased with the use of larger resin particles. This causes mass transfer resistance and lowers the column efficiency, because large molecules can only bind to the outer surface of the resin bead. Therefore, longer residence time is required to find binding ligands inside the resin particles, which in turn leads to a slow adsorption process. Since high throughput is very important for processing large sample volumes, the use of large particles, in particular for the adsorption of large proteins, has an impact on the overall productivity.
In sum, current chromatographic techniques cannot be easily scaled up to meet the demands in industry. The methods suffers from the drawbacks of being time consuming and expensive to practice in industrial scale.
Accordingly, there is a need to provide alternatives to column chromatography. Methods alternative to chromatography include membrane filtration, aqueous two-phase extraction, precipitation, crystallization, monoliths and membrane chromatography have been proposed (Przybycien et al “Alternative bioseparation operations: life beyond packed-bed chromatography” Curr Opin Biotechnol. 2004; 15(5):469-78).
Paril et al. J. Biotechnol. 2009; 141: 47-57 provide a means and methods for adsorption of pDNA on microparticulate charged surface. Specifically, Paril et al. uses polystyrene based microparticles provided by Rohm and Haas to adsorption of pDNA. However, Paril et al. does not teach how to prepare these microparticles such that they were able to adsorb pDNA. Generally, microparticles refer to particles having micron size.
There is still a need to provide an alternative, preferably improved methods for obtaining biomolecules from a fluid alternative to standard chromatographic techniques. The technical problem of the invention is to comply with one or more of the above mentions needs.